CN101330934B

CN101330934B - A moldable biomaterial for bone regeneration, preparation method and uses thereof

Info

Publication number: CN101330934B
Application number: CN2006800469268A
Authority: CN
Inventors: 米夏埃多·西德勒; 克劳泽·黑勒布兰德; 安德烈斯·许茨; 萨布里纳·克吕格尔
Original assignee: Scil Technology GmbH
Current assignee: Bionet Pharma GmbH
Priority date: 2005-12-14
Filing date: 2006-12-14
Publication date: 2013-03-20
Anticipated expiration: 2026-12-14
Also published as: CN101330934A; AU2006326271B2; EP1960008A2; WO2007068489A3; AU2006326271A1; US20090148487A1; KR20080081290A; JP2009519065A; WO2007068489A2; KR101105890B1; JP5068269B2

Abstract

The present invention is directed to a moldable biomaterial comprising a particulate solid porous material and a biodegradable paste material. The paste material and the particulate solid porous material form a matrix usable for the replacement or augmentation of bone. In various embodiments the matrix has a high structural integrity, which does not immediately or shortly after implantation collapse into an amorphous non-porous mass, maintains its porosity after implantation, shows biphasic degradation after implantation and/or has a good resistance against being washed out when it is applied to a wet opened implant site. Active agents can be incorporated in the moldable biomaterial of the present invention, such as bone growth factors. Kits, implants, method of manufacturing as well as medicinal uses are also provided.

Description

A kind of moldable biomaterial for osteanagenesis and its production and use

Technical field

The present invention relates to a kind of moldable biomaterial, it comprises microgranular solid porous material and biodegradable paste material.

Described paste material and microgranular solid porous material are formed for the substrate of bone replacement or bone lengthening.In various embodiments, described substrate has the structural intergrity of height, can at once or not subside in the short time after the implantation and be the non-porous substance without definite form, and after implantation, keep porous, during the transplantation site of the opening that shows two stage degraded after implanting and/or be applied to wet when this substrate, has the good anti-ability of washing out.

Activating agent, for example skeletal growth factor can be added in the moldable biomaterial of the present invention.

The present invention also provides kit (kit), implant, preparation method and medical application.

Background technology

Spinal fusion or spinal arthrodesis (for example, lumbar spinal column fusion) are usually as causing chronic lower pain in the lumbar region pain patient " final means " to use by the spine regression with shakiness.The method that is used for the treatment of a kind of suggestion of the lumbago that is caused by the joint deformity of intervertebral disc cracking or regression, disc collapses and regression spinal joint is to remove intervertebral disc and replaced by the device of porous, and it allows the growth of bone and the fusion of adjacent vertebrae.This integration technology comprises front road lumbar intervertebral fusion (ALIF), posterior lumbar interbody fusion (PLIF), the intervertebral spinal fusion approach lumbar intervertebral fusion (TLIF) except Posterolateral fusion, in Posterolateral fusion, fusing device is placed on the rear outside rather than replaces intervertebral disc.

The autogenous bone graft that is derived from crista iliaca is the golden standard material for spinal fusion.But using ileum is because patient has produced other problem as obtaining on a declining curve from the position of body bone.These problems comprise conventional postoperative wound pain, because may form Complex regional pain disease that neuroma causes, infection, postoperative local hematoma, to the osteoarticular damage of sacrum, to damage and soft tissues of pelvis's problem of pelvis ligament.In addition, autogenous bone graft has limited availability and inconsistent bone mass.Therefore, the use of autograft will be replaced by the method for using the bone substitute binding growth factor, and described somatomedin for example is those somatomedin of the TGF-β that comprises BMP-2, BMP-7 and GDF-5/BMP family.

These factors are used together in conjunction with following substances: collagen, collagen and carboxymethyl cellulose, and such as OP-1 (Osigraft

(OP-1 implant)/OP-1 plaster), Infuse (InductOs

), Collagen/hydroxyapatite, calcium phosphate binding agent (Bone source

), ceramic bovine bone, β-TCP, β-TCP/ hydroxylapatite compound (TCP/HA 15: 85, TCP/HA 40: 60), β-TCP polymer composites, it comprises the PLA-DX-PEG gel copolymer, or hydrogel.

WO94/15653 discloses the prescription that comprises tricalcium phosphate (TCP), TGF-β and collagen.TCP has been disclosed the carrier that transports as TGF-β.

EP1150726 has described the skeletonization spongy body that is used for inducing new bone growth, and it comprises can resorbent cavernosa material, osteogenic factor and mineral microparticle.

In PCT/EP2005/006204, the present inventor provides a kind of pastel of original position sclerosis, it comprises plasticizer, water-fast polymer and water-fast solid filler, and optional pore former, described pore former contact afterwards hardening with waterborne liquid such as water or body fluid.

But there are many shortcomings in traditional fusing device or biomaterial, and for example, they are not withstand voltage, need the bone growth promoting substance of unphysiological high concentration, and collagen-based material is as described supervened the risk of unwanted side effect.Other complex (for example, binding agent) immediately or collapse in the short time without the non-porous substance of determining form, and can not keep the physical integrity of porous matrix after implantation.Biodegradable implant material such as the bata-tricalcium phosphate granule of describing among the WO03/043673 or HA nanoparticle suspension thing, when being used to the position of moistening opening, during such as the environment of severe haemorrhage, is easy to be washed out or cracked.Biomaterial such as hydroxyapatite is non-biodegradation or part biological degraded, and it keeps in vivo for a long time.

Another limiting factor of hardening material is time range short between hardening process and the application and lacks porous (referring to for example classical calcium phosphate binding agent (CPC), for example Biobon

(α-BSM, US2005/0089579), Biocement D and H, Biofill

, Bonesource

, Calcibon

, Cementek

, Mimics Biopex And Norian

SRS

More example has description in PCT/EP2005/006204, the document is all incorporated this paper by reference into).Most of these obtainable CPC prescriptions are to be grouped into by two kinds of one-tenth that can react also hardening when mutually mixing.Before application immediately with pulverous composition and aqueous solution to form injectable pastel, some aqueous solution comprises accelerator or promoter.These paste compositions are difficult to preserve with the pasty state viscosity and surpass several hours to a week or many weeks and not hardening in most of the cases, even surpasses 20 minutes to about 60 minutes or reaches about 15 minutes, and this depends on the temperature that self-curing reacts occurs.Comprise that these contain the CPC of decalcified bone matrix (DBM), for example in US2005/0084542, describe, use two kinds of inorganic constituentss to carry out bone cement reaction (cement reaction), after adding physiological aqueous fluids, the support that is used in the body forms.

Another shortcoming of premixed pasty state prescription is necessary aseptic preparation, because use conventional final sterilization method, such as the gamma sterilization, it is impossible that final products are sterilized.Therefore, destruction that reason is activating agent.Thereby manufacturing is meticulous and expensive.

Summary of the invention

In a word, although the biomaterial such as the ceramic material is arranged, such as β-TCP, hydroxyapatite or both mixture, bone cementum, comprise the material based on polymer, or the as described above existence of collagen, but still have further improvement biomaterial and be used for comprising that spinal fusion, cranium jaw face reproduce, the indication of joint reconstruction and fracture repair improves the demand of the method for bone lengthening.Existence is to the demand of the compositions of raising biocompatibility and biodegradability, and infiltration and migration that described compositions provides porous support to be used for cell in vivo replace biomaterial to pass through the bone structure, have alleviated simultaneously the burden of body.Preferably, described compositions should be biomaterial or the device that has overcome aforesaid one or more shortcomings that traditional fusing device in the prior art or biomaterial exist.

Another purpose provides a kind of compositions of improving biocompatibility and biodegradability, and described compositions is adjusted to defect, and provides in vivo porous support to be used for cellular infiltration and bone replacement.

Another object of the present invention provides the biomaterial of original position sclerosis, and it is suitable for being implanted into the curee who needs bone lengthening by the compositions that can form the macropore support after putting into defect, and described biomaterial hardens in vivo.

Another object of the present invention provides a kind of biomaterial of original position sclerosis, it is suitable for being implanted into the curee who needs bone lengthening by the compositions that can form the macropore support after putting into defect, described biomaterial hardens in vivo, and wherein moldable biomaterial is not the binding agent of calcic.

Another object of the present invention provides a kind of original position sclerosis biomaterial, it is suitable for being implanted into the curee who needs bone lengthening by the compositions that can form the macropore support after putting into defect, described biomaterial hardens in vivo, and has porous and/or the mechanical strength of improvement.

Another object of the present invention provides a kind of original position sclerosis biomaterial, it is suitable for being implanted into the curee who needs bone lengthening by the compositions that can form the macropore support after putting into defect, described biomaterial hardens in vivo, and it has been easy to prepare and show the storage stability of improvement.

Another object of the present invention provides a kind of biocompatible and biodegradable compositions of improvement, and the sustained release activating agent.

Another object of the present invention provides a kind of biocompatible and biodegradable compositions of improvement, and it is suitable for comparing the more transfer system of the activating agent of low dosage with traditional device as permission.

Another object of the present invention provides a kind of being designed for such as long bone and merges or the bone of the improvement that the bone of spinal fusion merges is transplanted the substituent material.

Another object of the present invention provides a kind of spinal implant, and it comprises a kind of bone growth promoting composition, merges to promote the bone between the adjacent vertebrae.

Another object of the present invention provides a kind of bone of the improvement for bone lengthening and transplants the substituent material, and described bone lengthening comprises maxillary surface bone lengthening and periodontal regenerative.

Surprisingly, the present inventor can provide a kind of moldable biomaterial that can realize these purposes and for the production of the correlation method of described biomaterial.

So the present inventor provides a kind of moldable biomaterial, it comprises having granular solids porous material and the biodegradable paste material that mean diameter is 100-4000 μ m.

Described paste material and solid particle porous material form a kind of substrate, and it is particularly advantageous in replacement or the increase of bone.The integrity at least about two to three day time in maintenance its structure of described substrate after transplanting, and after implanting the physiological environment that the bone replacement occurs, keep its loose structure." the structure property finished " refers to substantially keep shape and the size of implanted substrate.This is owing to described two kinds of composition systems, and wherein said solid particle porous material forms has the structure of high mechanical properties, and described paste material keeps together the solid particle porous material.

The structural intergrity of the moldable biomaterial of the present invention with compare such as other pastel of prior art compositions ceramic or the Nanocrystalline Hydroxyapatite float, the structure of described prior art pastel is subsided immediately or at short notice after implantation and is non-porous substance without determining form.Being also advantageous in that of the substrate of moldable biomaterial of the present invention keeps its porous after implantation, this is very important for bone replacement or bone lengthening process.

Moldable biomaterial of the present invention is a kind of pair of composition system, and it is shown as the degraded of two stages in vivo after transplanting, that is, each component, the granular solids porous material forms different degradation kineticss with biodegradable pastes.Because described two stages degraded, moldable biomaterial of the present invention keeps loose structure after implantation, to improve bone formation.In addition, described two stages degraded can make sustained release or the transportation that improves such as the activating agent of bone growth inducing agent.

Preferably, the moldable biomaterial of the present invention has the two stage degradation models of one of this component, and it finally becomes three stage degradation models of bicomponent system after implanting.Described three stage degradation models can be respectively caused by the different degradation kinetics of the ceramic component of the polymers compositions of granular solids porous material, biodegradable paste material and biodegradable paste material.

An advantage of moldable biomaterial of the present invention is that it has moldable bonding denseness, and the position of use is admitted and is retained at the position that it is easy to be used.With compare such as other biodegradable implant material of bata-tricalcium phosphate granule or HA nanoparticle suspension thing, when implant of the present invention is used for the implant site of moistening opening, during such as the severe haemorrhage environment, it has the well anti-ability that washes out.

Another advantage of the present invention is, organic solvent to the negative effect that is included in the active substance in the embedded material can be by will comprising organic solvent biodegradable paste material be eliminated with comprising to separate such as the active substance of the granular solids porous material of bata-tricalcium phosphate granule.

Another advantage of the present invention is, compare with traditional paste composition, but the pressing mold biomaterial has increased porous, has reduced polymer content, thereby has alleviated the burden of body.In addition, compare with traditional paste composition, the mechanical stability of moldable biomaterial has strengthened.

The kit of two kinds of independent components by the moldable biomaterial of a kind of the present invention of comprising is provided makes two kinds of components be mixed into immediately before use possibility.Before using moldable biomaterial of the present invention, the active substance that will contain immediately ceramic material mixes with the biodegradable pastes that contains organic solvent, compare with the prescription that contains organic solvent and activating agent under being in condition of storage, further increased the pot-life such as the activating agent of bone growth inducing albumen.

Another advantage of kit is that because two kinds of components are separated, biodegradable paste material can carry out final sterilization, for example sterilizes by gamma.

One aspect of the present invention is, with the polymer pasty state phase ratio such as the pastel among the PCT/EP2005/006204, although the polymer content of embedded material has reduced, but surprisingly, not add the hardness of polymer pasty state thing of porous ceramic film material high 2.5 times for the hardness ratio that showed after 2 hours of described implant material.

Other effect of the present invention or advantage will be described below.

Embodiment of the present invention are:

(1) a kind of moldable biomaterial, it comprises:

A) mean diameter is the granular solids porous material of 100-4000 μ m, and

B) biodegradable paste material.

(1a) preferentially, described granular solids porous material forms the internal structure of described moldable biomaterial, and consists of mechanical strength, and described biodegradable paste material is in together the granular solids porous material.Described biodegradable paste material further strengthens the mechanical strength of solid particle porous material.Because the present invention, the granular solids porous material is merged in the single structure the preceding, and described structure has, with the solid particle porous material (for example, free-pouring ceramic particle) compares improved mechanical property with biodegradable paste material.

(1b) more preferably, described moldable biomaterial is that a kind of bone replaces material.

(1c) further preferably, described moldable biomaterial is anhydrous.

(1d) most preferably, described biodegradable paste material comprises non-collagen based polyalcohol.

(1e) in another embodiment, described biodegradable paste material comprises a kind of synthetic polymer.

(1f) in another embodiment, the polymer content of described moldable biomaterial is less than 35wt%, preferably is less than 25wt%, is less than 15wt%, most preferably from about 10-15wt%.

(1g) in another embodiment, the granular solids porous material of described moldable biomaterial and the content of water-insoluble solid filler surpass 50wt%, preferably surpass 55wt%, most preferably from about 58-62wt%.

(1h) in another embodiment, the amount of the solid material in the moldable biomaterial is at least 55wt%, preferably between 55wt% to 80wt%, between 55wt% to 70wt%, between 55wt% to 65wt%, between 58wt% to 62wt%.Solid material in the preferred moldable biomaterial and/or solid particle porous material are selected from calcium sulfate, calcium phosphate and are derived from the bone graft substitute material of cattle.

(2) the moldable biomaterial of embodiment 1, wherein

Described biodegradable paste material is a kind of pastel, and it comprises:

I. plasticizer, it is biocompatible organic liquid water-soluble or that can mix with water;

Ii. water-fast polymer, it dissolves in described plasticizer, and is biocompatible, biodegradable, and/or can be biological resorbent; And

Iii. water-fast solid filler, it is insoluble to described plasticizer,

Wherein said pastel is preferably injectable.

(2a) the moldable biomaterial of embodiment 1, wherein

A) described granular solids porous material comprises by calcium phosphate or calcium sulfate, preferably phosphoric acid DFP, the most preferably granule made of bata-tricalcium phosphate, preferably ceramic granule, its mean diameter are 100-4000 μ m, 100-3000 μ m, 100-2000 μ m, 100-1500 μ m, 500-4000 μ m, 500-3000 μ m, 500-2000 μ m, 500-1500 μ m, or 500-1000 μ m, and

B) described biodegradable paste material is a kind of pastel, and it comprises:

Iii. water-fast solid filler, it is insoluble to described plasticizer,

Wherein said pastel is preferably injectable on the one hand.

(2b) randomly, described biodegradable paste material is injectable and is stable in its packing, and in case with after aqueous media or body fluid contact, can harden to form the solid implant at former position.

(2c) described granular solids porous material of the present invention be biodegradable, can be biological resorbent, and/or biocompatible, preferred biomaterial macropore and/or micropore, it has bone conductibility, and the activating agent or its conjugate that add such as bone growth promoting substance have osteoconductive nature equally.It can increase the mechanical stability of moldable biomaterial, and at the b such as component of polymer) in after the biodegradable paste material degraded, stay as the substrate that is used for the replacement of cellular infiltration and bone subsequently.

Preferably, described solid porous material has interconnective hole.

Preferably, described granular solids porous material is a kind of inorganic calcium chemical compound or such as the silica-based materials of bio-vitric.More preferably, described granular solids porous material is calcium phosphate, tricalcium phosphate most preferably, bata-tricalcium phosphate, type alpha tricalcium phosphate, apatite, the binding agent that contains calcium phosphate, tetracalcium phosphate, two stage tricalcium phosphate/hydroxyapatite material (TCP/HA) or its compositions or mixture, most preferably bata-tricalcium phosphates.

Preferably, described granular solids porous material has the graininess outward appearance, more preferably as free flowing granule.The preferred mean diameter of described granular solids porous material and preferred embodiment thereof are 100-4000 μ m, 100-3000 μ m, 100-2000 μ m, 100-1500 μ m, 500-4000 μ m, 500-3000 μ m, 500-2000 μ m, 500-1500 μ m, or 500-1000 μ m.

In addition, described granular solids porous material is optional to be the carrier of activating agent, as hereinafter describing in the embodiment 6.Preferably, described activating agent is equably at least part of or be distributed in equably on the granular solids porous material.Most preferably, described granular solids porous material evenly or at random be coated with activating agent such as morphogenetic proteins, it includes but not limited to BMP-2, BMP-7 or GDF-5.The described activating agent of BMP-2, BMP-7 or GDF-5 that comprises can use by activity form well known in the prior art, the maturation protein or bioactive fragment or its variant (the mature human BMP-2 albumen that for example, extends with the N-terminal alanine) that comprise them.

(2d) the described water-fast solid filler in one embodiment the described biodegradable paste material comprises:

A) a kind of inorganic compound, and/or

B) a kind of organic compound.

Inorganic compound in this embodiment preferably calcium compounds, magnesium oxide, magnesium hydroxide, from, magnesium carbonate, silicon dioxide or its compositions or mixture, more preferably calcium sulfate, calcium carbonate or calcium phosphate, most preferably tricalcium phosphate, bata-tricalcium phosphate, type alpha tricalcium phosphate, apatite, the binding agent that contains calcium phosphate, tetracalcium phosphate, two stage tricalcium phosphate/hydroxyapatite material (TCP/HA) or its compositions or mixture.

Described organic compound comprises chitosan, collagen, calcium alginate, poly-(2-hydroxyethyl methylacrylate), hyaluronic acid or derivatives thereof, cellulose or derivatives thereof, or the starch or derivatives thereof.

In the compositions of one or more chemical compounds of mentioning in (2d) is also included within.

Randomly, biodegradable paste material comprises at least a other water-fast solid filler that contains calcium, is preferably selected from calcium sulfate, calcium carbonate, calcium hydrogen phosphate or hydroxyapatite.

(2e) the described water-fast polymer in the biodegradable paste material in embodiment comprises poly-(alpha-hydroxy acid), poe, poly-anhydride, polyamino acid, polyglycolic acid (PGA), polylactic acid (PLLA), poly-(D, L)-lactic acid (PDLLA), poly-(lactic acid-altogether-hydroxyacetic acid) (PLGA), poly-(lactic acid-altogether-hydroxyacetic acid) Polyethylene Glycol (PLGA-PEG) copolymer, poly-(3-hydroxybutyrate) (P (3-HB)), poly-(3-hydroxypentanoic acid) (P (3-HV)), poly-to dioxanone (PDS), gather (6-caprolactone) (PCL), poly-anhydride (PA) poe, polyglactine, or its copolymer, terpolymer, block copolymer, compositions or mixture.

Preferably, described water-fast polymer is PLGA, and the ratio that preferred water-fast polymer has lactic acid/hydroxyacetic acid is less than 75: 25, preferred 50: 50.

Equally preferably, described water-fast polymer is terminated polymer.Terminated polymer comprises modification, but does not contain the free carboxyl group end group, and with the polymer phase ratio of non-end-blocking, it causes the variation of polarity.

Preferably, the described water-fast polymer polymer that is non-end-blocking or with the polymer of free carboxyl group end group.With the polymer phase ratio of end-blocking, such polymer can be better with polarity, the activating agent of preferred positively charged interacts.This has just produced with the polymer phase of end-blocking than the advantage of sustained release better.

On the one hand, the content of the water-fast polymer of described biodegradable paste material is equal to or less than 40wt%.

In another embodiment, the density of described biodegradable paste material compositions is equal to or greater than 1.21g/ml, preferably between 1.3g/ml to 1.5g/ml.

(2f) the described plasticizer in the described biodegradable paste material in embodiment comprises Polyethylene Glycol (PEG) 400, PEG200, PEG300, PEG600,1, the 3-butanediol, Oleum Ricini, METHYLPYRROLIDONE, 2-Pyrrolidone, C2 to C6 alkanol, propylene glycol, solketal, acetone, methyl acetate, ethyl acetate, ethyl lactate, methyl ethyl ketone, dimethyl formamide, dimethyl sulfoxine, dimethylsulfone, oxolane, decyl methyl sulfoxide (decylmethylsulfoxide), oleic acid, Allyl carbonate, N, N-diethyl-m-toluamide, 1-positive dodecyl aza cyclohepta alkane-2-ketone or its mixture.

Preferably, the plasticizer in the described biodegradable paste material comprises Polyethylene Glycol (PEG) 400.

Preferably, the plasticizer content of described biodegradable paste material is 40-95wt%, more preferably 40-55wt%.

(2g) in described biodegradable paste material, preferred 1: 1 to 5: 1 of the ratio (being weight ratio) of water-fast solid filler and water-fast polymer, more preferably 1: 1 to 3: 1, more preferably 1.5: 1, be less than 50wt% as in a mixture, containing, the water-fast solid filler of preferred 30-36wt% and be less than 40wt%, the water-fast polymer of preferred 20-25wt%.

(3) embodiment 1 or arbitrary moldable biomaterial of 2, it has moldable viscosity, and preferably it is preferably solid porous implant in case with after aqueous media or body fluid contact, can harden to form the solid implant in former position.

(3a) more preferably, the moldable biomaterial of any in the above-mentioned embodiment, wherein said solid implant has interconnective hole.

(4) any moldable biomaterial among the embodiment 1-3, wherein, component a) and b) use according to a certain ratio, to form adhesive product, preferred proportion is 1: 0.3wt% to 1: 2wt%, preferred 1: 1wt% to 1: 2wt%, more preferably 1: 1.3wt% to 1: 1.7wt%, most preferably 1: 1.4 to 1: 1.6wt%.

In a preferred embodiment, the structure of moldable biomaterial be a) and b) two components system.The granular solids porous material strengthens the mechanical strength of system in vivo after the hardening, and biodegradable paste material provides bonding semi-solid structure, and it makes the granular solids porous material and keeps together before application and in using.After being applied to transplantation site, described semi-solid jointing material hardens, and by being formed to small part solid bridge between the granule in vivo solid porous granule is connected together.Therefore, the combination by two kinds of components has produced bonding moldable material, and is different from the granular materials such as β-TCP, and described moldable material is the fixing biomaterial in a kind of part.This bonding moldable material will be transformed into for cellular infiltration and osteoplastic bonding support subsequently after the sclerosis of former position in aqueous solution or body fluid.The moldable characteristic of biomaterial be conducive to fill various devices or such as bone material fill, critical size is damaged, bone formation in the various application of bone defect and spinal fusion.Preferably, adopt a) and b) ratio be 1: 0.3wt% to 1: 2wt%, preferred 1: 1wt% to 1: 2wt%, more preferably 1: 1.3wt% to 1: 1.7wt%, most preferably 1: 1.4wt% to 1: 1.6wt%.These ratios allow the ceramic granule to be combined into ideally binding system, and vice versa, and acquisition is used for the final embedded material with maximum porosity such as the indication of spinal fusion.Paste material in the final moldable biomaterial and the ratio between the ceramic particle are regulated the total porosity of the biomaterial after the sclerosis of former position, and avoid material subside to promote regenerative process.Even after polymers compositions degraded, the porous support of granular solids porous material still is retained in application site, its tissue that will newly be formed replaces such as bone or cartilage subsequently.

(5) the moldable biomaterial of any, wherein components b among the embodiment 1-4) pastel comprise water-soluble degraded regulator.

(5a) in one embodiment, the described water-soluble degraded regulator in the moldable biomaterial comprises one or more in the following substances:

(a) sweller, the preferred cellulose derivant;

(b) surfactant, the block copolymer of optimization ethylene oxide and expoxy propane is such as Pluronics Or Tween

80; Or

(c) porogen, such as trehalose, mannitol, sucrose, Sorbitol, physiological aminoacid, for example glycine, glutamine, arginine, sodium citrate, sodium succinate and sodium phosphate, sodium chloride, polyvidon (PVP), solid PEG are such as PEG4000, PEG10000, sodium bicarbonate, calcium sulfate or chitosan; Or

(d) gas or gas form agent, such as calcium carbonate or sodium bicarbonate.

(5b) content of the water-soluble degraded regulator in the biodegradable paste material is less than 10wt%, preferably be less than 5wt%, more preferably 1-4wt%, further preferred 1.5-3.5wt%, 2-3.5wt% most preferably is based on components b) the gross weight of pastel.

(5c) carboxymethyl cellulose preferably of the water-soluble degraded regulator in the biodegradable paste material, more preferably carboxymethyl cellulose is less than 10wt%, preferably be less than 5wt%, more preferably 1-4wt%, further preferred 1.5-3.5wt%, 2-3.5wt% most preferably is based on components b) the gross weight of biodegradable paste material.

(5d) mean diameter of the water-soluble degraded regulator in the biodegradable paste material is preferably less than 1000 μ m, preferred 25-1000 μ m, more preferably 50-500 μ m, 100-300 μ m most preferably, the viscosity that preferably has 1500-2500mPa*s, preferably has 0.2 to 1.3 substitution value, more preferably 0.6 to 1, most preferably from about 0.7.

(6) the moldable biomaterial of any among the embodiment 1-5 further comprises

C) activating agent, the activating agent of preferred therapeutic effective dose, most preferably, described activating agent is a kind of tissue regeneration agent, skeletal growth factor, osteoinductive agent or chondrocyte induction agent.

(6a) the activating agent preferred coated in the moldable material is on the granular solids porous material or be in the solid particle porous material.

(6b) on the other hand, described activating agent is coated on the water-fast solid filler or is dissolved or suspended in the plasticizer, preferably is coated in equably in the water-fast solid-filling agent of biodegradable paste material.

(6c) preferably, not containing moldable biomaterial activating agent or that preferably contain activating agent has the body internal skeleton and induces and/or bone conduction cartilage or periodontal ligament reproducing characteristic.

(7) the moldable biomaterial of any among the embodiment 1-6, wherein said activating agent is selected from hormone, cytokine, somatomedin, preferred skeletal growth factor, antibiotic and micromolecule.

(7a) on the one hand, described activating agent is parathyroid hormone (PTH) and/or PTH 1-34 peptide.

(7b) on the other hand, described activating agent is that bone is induced or chondrocyte induction albumen.

(7c) on the other hand, described activating agent be TGF-β family or BMP or GDF family, be preferably selected from BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15 or BMP-16; GDF-1, GDF-2, GDF-3, GDF-4, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10 or GDF-11.If be fit to, in this respect, the compositions of two or more of these activating agents also is included.

(7d) on the other hand, described activating agent is the cartilage derived tretinoin sensitive Protein of regenerating bone or cartilage (CD-RAP).

(7e) preferably, described activating agent is selected from BMP-2, BMP-7 and GDF-5.

(8) the moldable biomaterial of any among the embodiment 1-7, every milliliter of biomaterial contains 5 μ g-2mg activating agents, preferred every milliliter of 250 μ g-2mg, most preferably every milliliter of 250 μ g-1mg.

(9) the moldable biomaterial of any among the embodiment 1-8, it shows the degraded of two stages of original position.

One of advantage of the present invention is that polymer and fixed granular solids porous material form compositions substrate, and described substrate is particularly advantageous in bone and replaces or bone lengthening.After about two to three days, during keep physiology's integrity of described substrate, depolymerization has increased, and in the time that surpasses several weeks, in the environment of the generation that bone replaces that biomaterial is newly formed, keeps the matrix structure of solid porous pottery.

Term " two stage degraded " refers to the degraded in two stages, initial stage degraded and second degradation period of polymer, and wherein the solid particle porous material will be absorbed such as osteoclast by for example cell again, and the bone that is newly formed replaces.Described the second degradation period can allow the further release of activating agent to accelerate remodeling process.This degradation model produces the release mode that can be divided into the different or continuous release stage.Such release stage can be for example discharged by the initial stage, and the further release during degraded and/or the final release when decomposing from polymer diffusion and polymers compositions form.

(10) the moldable biomaterial of any among the embodiment 1-9, described biomaterial keep physical integrity in time of 2-3 days at least after the sclerosis of former position, and/or keep the porous particle structure after the polymers compositions degraded.

(10a) the moldable biomaterial in preferred embodiment comprises:

(a) bata-tricalcium phosphate

(b)i.PEG?400

ii.PLGA

Iii. calcium phosphate is selected from the binding agent, calcium carbonate, hydroxyapatite, calcium hydrogen phosphate, bata-tricalcium phosphate and the type alpha tricalcium phosphate that contain calcium phosphate, or its mixture; And

Iv. sanlose randomly.

(10b) the moldable biomaterial in the further preferred embodiment comprises:

(a) mean diameter is the β-TCP granule of 500-1000 μ m, and preferred total porosity is 20-70%;

(b) i.PEG 400:40-50wt%, preferred 40-45wt%;

Ii.PLGA:20-25wt%, preferred 22-25wt%;

Iii. calcium phosphate is selected from the binding agent and the bata-tricalcium phosphate that contain calcium phosphate: 25-40wt%, preferred 30-35wt%; And

Iv. sanlose randomly.

Embodiment (10a) and (10b) in optional sanlose constituent content preferably be less than 10wt%, preferably be less than 5wt%, more preferably 1-4wt%, 2-3.5wt% most preferably is based on components b) the gross weight of pastel.

Total porosity according to the present invention refers to macroporsity and/or the micro-porosity such as the synthesising biological material of β-TCP.Porosity can pass through method known to those skilled in the art, measures such as mercury porometer and micro-CT method.

Preferably, β-TCP is pure phase β-TCP, to avoid undesirable side effect in the biomaterial degradation process.Phase purity can be passed through such as Tadic and Epple, and (2004), the methods such as the high-resolution X-ray diffraction method of describing among the Biomaterials 25:987-994 are measured.

(11) kit, it comprise as the component of implementing the isolation of the moldable biomaterial of any among the scheme 1-10 a) and b), or as the scheme of enforcement 6-10 in any moldable biomaterial isolation component a), b) and c).

Because the present invention, two kinds of components a) and b) isolated, a), b) and c) isolated, or b) and c) isolated, improved activating agent stability in time, thereby strengthened the regeneration potential of moldable biomaterial.This is conducive to the economic validity of for a long time storage and final products.

In addition, can be by using one or more primary package assemblies, such as blister, vial, further prolong the stability of paste material, avoid the water absorption or diffuse into biodegradable paste material, described package component is generally used for the medicine preparation and is well known to those skilled in the art.Compare with the product (for example single component product) of preparing to use, two kinds of another isolated advantages of component are, the industry preparation of moldable biomaterial (has for example been simplified significantly, pass through final sterilization), and with other industrial production process, compare such as aseptic preparation technology, cost.

(11a) in a preferred embodiment, described kit also can comprise the equipment of use, for example, and syringe, medicator, filling gun, attachment device, spinal fusion equipment, Wicresoft's application apparatus, accent spatula, crucible, or its combination.

(12) a kind of implant, it comprise as the component of implementing any described moldable biomaterial among the scheme 1-10 a) and b), or as the component of implementing any described moldable biomaterial among the scheme 6-10 a), b) and c), the implant of preferred hardening namely obtains described hardening implant in case contact with aqueous solution.

(13) a kind of method for preparing moldable biomaterial, it comprises that with a kind of pastel and mean diameter be the particle porous material mixing of 100-4000 μ m, the particle porous material described in the preferred above-mentioned embodiment, wherein said pastel comprises

I. plasticizer, it is water-soluble or the biocompatibility organic liquid that mixes of Yi Yushui;

Ii. plasticizer, it is water-soluble or the biocompatibility organic liquid that mixes of Yi Yushui;

Iii. water-fast solid filler, it is insoluble to described plasticizer,

So that mixture has moldable viscosity, in a single day it contact with aqueous media or body fluid to harden in position and form solid porous implant.

(14) method of embodiment 13, wherein with described biodegradable paste material drying to reduce water impurity and/or to adopt water-free component i, ii and/or the iii preparation.

The advantage of preparation process is, has further increased the stability of pastel and each moldable biomaterial, and for example the polymer chemistry of sclerosis, moldable biomaterial transforms or chain interruption to avoid too early.

(15) application of any described moldable biomaterial among the embodiment 1-10, the application of the kit of embodiment 11, or the application of the implant of embodiment 12, for the preparation of pharmaceutical composition or armarium, to be used for various indications, for example, spinal fusion, bone defect, critical size is damaged, nonunion, the joint relocates, and preferred knee or buttocks relocate, fracture repair, repair of cartilage, full depth or segment thickness are damaged, maxillary surface reproduces, sinus highmori promotes art, periodontal is repaired, periodontal disease, the lumbar intervertebral disc degeneration disease, spondylolisthesis disease, osseous tissue is filled.

(15a) preferably, described pharmaceutical composition or armarium are used for merging adjacent vertebra.In this embodiment, pharmaceutical composition or armarium preferably are inserted between the adjacent vertebra, randomly in the spinal implants such as spinal fusion cage device or partition.

The spinal implants that is used for operation on vertebra is well known to those skilled in the art, and with various profile use, described profile comprises cylinder or conical cage (screw thread cage), box-like or rectangle cage (non-threaded cage), Horizontal Cylinder (for example BAK cage), perpendicular rings (for example Harms cage), open box (for example Brantigan cage), solid rectangle parallel tubular shape partition, for example LT-Cage Lumbar Tapered Fusion Device, INTER FIX ^TMWith INTER FIX ^TMThreaded Fusion Devices, and can biological resorbent cage, for example with or do not have a Telamon Peek of pedicle screw ^TMWith Telamon Hydrosorb ^TMAnd fixing device (spinal fusion progress (Advances in spinal fusion), molecular science (MolecularScience), biomechanics (Biomechanics) and Clinical Processing (Clinical Management), Marcel moral gram (Marcel Dekker), Inc NewYork 2004).Above further described different integration technologies, and be well known to those skilled in the art.

Preferably, the moldable biomaterial in the above-mentioned embodiment is filled in the spinal implant so that described Material Filling the structure of space or hollow, form rather than bone formation to avoid fibrous tissue.Randomly, the described implant that is filled can be impregnated in the short time before being applied to health or tissue, soak or moistening in waterborne liquid, body fluid or sodium chloride solution, thereby formation is for the porous support of cell migration and tissue regeneration the best.

Perhaps, described pharmaceutical composition or armarium also can be used for being with or without an inner fixing sections or the rear outside fusion of multi-segmental.In this embodiment, described pharmaceutical composition or armarium preferably are embedded into the rear outside of vertebra, and the optional inside that is with or without is fixed.

(15b) this embodiment has been considered among the embodiment 1-10 any, the external member of embodiment 11, or the moldable biomaterial of the implant of embodiment 12 can be used in the spinal fusion method, the treatment bone defect, the treatment critical size is damaged, the treatment fracture, the treatment nonunion, treatment lumbar intervertebral disc degeneration disease, the reach for the treatment of bone vertebra, the treatment bone is damaged or merge in the method for adjacent vertebrae, be included between the adjacent vertebrae and embed among the embodiment 1-10 that any is described, the member of the kit of embodiment 11, or the moldable biomaterial of the implant of embodiment 12, described moldable biomaterial is positioned at the spinal implants such as spinal fusion cage or partition.

This embodiment has considered also among the embodiment 1-10 that any is described, the moldable biomaterial of implant external member or embodiment 12 of embodiment 11 can be used in bone and/or the chondrocyte induction method, comprises embedding among the embodiment 1-10 that any is described, the moldable biomaterial of implant member or embodiment 12 of the kit of embodiment 11.

(16) but by the pressing mold biomaterial of the method for embodiment 13 or 14 preparation.

(17) a kind of pharmaceutical composition, it comprises among the embodiment 1-10, and any is described, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11.

(18) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 is for the preparation of the application in the pharmaceutical composition of bone lengthening.

(18a) in a preferred embodiment, described bone lengthening is being followed wound, pernicious or manually damaged, or the prerequisite of implant is set subsequently.

(19) any is described among the embodiment 1-10, the application of the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 in the pharmaceutical composition damaged for the preparation of the treatment bone.

(19a) in a preferred embodiment, described bone is damaged to be that bone defect, critical size are damaged, nonunion, joint relocate, damaged, the maxillary surface after relocating such as knee joint and buttocks regional damaged or follow the bone of nipper apicoectomy damaged, the bone of excision, exodontia, defect of skull, brainpan or the viscerocranium of cyst or tumor is damaged, osteoporosis or operation remove residual tooth.

(20) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 for the preparation for the treatment of degenerations, wound lumbar intervertebral disc disease, spinal fusion, vertebral body fracture, vertebra is shaped and the pharmaceutical composition of kyphosis shaping in application.

(21) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 is for the preparation of the application in the pharmaceutical composition for the treatment of bone dehiscence.

(22) any is described among the embodiment 1-10, the application of the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 in the pharmaceutical composition that increases of the upper jaw bone that promotes art or atrophy for the preparation of the maxillary degree or lower jaw groove ridge.

(23) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 is for the preparation of the application in the pharmaceutical composition of the regeneration in cavity filling, the periodontal disease and/or the supporting guide tissue regeneration in the periodontal disease.

(24) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 is for the preparation of the application that promotes in the chondrogenetic pharmaceutical composition.

(25) any is described among the embodiment 1-10, the moldable biomaterial of implant kit or embodiment 12 of embodiment 11 is for the preparation of the application in the pharmaceutical composition of at least a cartilage disease for the treatment of.

Preferably, described osteopathia is selected from the following disease that relates to the bone marrow interstital stem cell cartilage differentiation: the articular cartilage damage that osteoarthritis, rheumatic arthritis, wound cause, osteochondral defect, through thickness or segment thickness are damaged, the reproducing of the keeping of the chondrocyte phenotypic characteristic in the body chondrocyte cell transplantation, ear, trachea or nasal cartilages, funny bone chondritis (osteochondritisdissecans), intervertebral disc or meniscal regeneration, fracture and/or be derived from the osteogenesis of cartilage.

The specific embodiment

Now with reference to as give a definition and the description of accompanying drawing of the present invention described in detail the present invention.

The definition of important technology term

In order to be conducive to understand principle of the present invention, the concrete syntax that will relate to some embodiment and describe these embodiments.Yet, be appreciated that to be intended to not consist of limitation of the scope of the invention that the change of the principle of the present invention of illustrating, other application and improvement can be expected, and often expect as the technical staff that the invention relates to the field herein.

The moldable biomaterial of A

Term " moldable biomaterial " refers to be easy to adopt any shape and form for example to fill the biomaterial in defect location or the hollow cavity that implants or hole.It comprises the compositions of suspension, dispersion or liquid, and preferred described compositions can be used or the injection use by Wicresoft.It also comprises plastic paste material.Preferably, described moldable biomaterial can harden under moist environment, preferably in human body or when contacting with human body fluid, that is, can harden at former position.Moldable biomaterial of the present invention is different from other traditional biomaterial, for example moldable CPC before the aqueous solution that uses such as saline solution or body fluid.With traditional self-hardening or spontaneous coagulation response composite, fully different such as apatite (PCA) the calcium phosphate implant material that adhesive composition or degree of crystallinity are lower, moldable biomaterial of the present invention preferably includes the granular solids porous material with skeleton property, rather than is used for one or more reactive components of Chemical Felter curing reaction.Preferably, described moldable biomaterial does not comprise decalcified bone matrix (DBM), preferred combination calcium phosphate.

Term " anhydrous " refers to that moldable biomaterial contains and is less than 5wt%, preferably is less than 3wt%, more preferably less than 2wt%, most preferably is less than the water of 1wt%, is measured by the method such as the karl Fischer method.Preferably, term " anhydrous " refers to only have in the moldable biomaterial that the Free water (for example unbound water) of trace exists.The minimizing of the content of Free water can reduce polymer, such as the degradation rate of PLGA, thereby prolongs pot-life of moldable biomaterial.

The water of trace refers to that the amount of water can not further reduce by metric system Preparation Method well known by persons skilled in the art, described method for example is method, the drying under reduced pressure of dry each component or heat up dry, in advance method, vacuum drying, the lyophilization of heat treatment component, if and be fit to, by molecular sieve and use and be used for packing to the packaging system with desiccant of the pharmaceutical preparation of moisture-sensitive.

Term " granule " such as granular materials, refers to the discrete solid particle of biomaterial, for example the powder of sand, grain or at least 1 μ m, preferably at least 50 μ m, most preferably at least 100 μ m.

Term " bonding " refers to stick together or stick together.It comprises that also the microgranular granule of at least part of for example particle porous material is connected to its adjacent granule formation bridge of small part by biodegradable paste material, so that the solid particle porous material keeps together.

Term of the present invention " sclerosis of former position " refers to, organic solvent be distributed to external environment and such as the organism of human or animal body or tissue in after, with aqueous media, as forming solid matrix after water, physiological solution or the body fluid contact.According to the application of indication and moldable biomaterial, such solid matrix also will comprise substrate, preferred implant, and described substrate has higher mechanical strength at least with after ambient body fluid contacts.

B granular solids porous material

That term " granular solids porous material " refers to is biodegradable, can be biological resorbent and/or biocompatible, preferred macropore and/or micropore biomaterial, it is bone conduction.It also refers to the solid material of fine particle, such as calcium phosphate.In the embodiment hereinbefore more detailed description is arranged.

The biodegradable paste material of C

As previously mentioned, the present invention generally provides a kind of biodegradable paste material that comprises following at least three kinds of components: plasticizer, and it is water-soluble or the biocompatible organic liquid of Yi Yushui mixing; Water-fast polymer, it is biocompatible, biodegradable and/or is can be biological resorbent and dissolve in the described plasticizer; And water-fast solid filler, it is insoluble to described plasticizer, and wherein said pastel is preferably injectable and be stable in its packing, and is being placed on the after-hardening of damaged place.

Preferably, the stability of premixed biodegradable paste material in packing continues several weeks at least, more preferably some months, most preferably at least one year.Stability can be regarded as the viscosity of each premix and moldability along with the time does not have significant change.Comprise normally used waterproofing packing, as be generally used for the packing that the non-intestinal in the medicinal application is used.

That the term that uses among the present invention " pastel " refers to is soft, mixture level and smooth, thickness or material, or the pastel of accessible preferred use syringe or Wicresoft's application (namely, can be by 16-to 18-number (gauge) syringe), it comprises at least three kinds of components, preferred at least four kinds of components are described in this description.Preferably, described biodegradable paste material should be compatible with activating agent, to avoid unnecessary degraded and/or the inactivation of activating agent.At least in some embodiments, described pastel is suspension, dispersion or liquid.

In a preferred embodiment, biodegradable paste material of the present invention and moldable biomaterial do not contain the toxicity material.Preferably, these toxicants have been avoided in process of production, because the extra cost that their production requirement is produced by the required removal step in the production process, and requirement is used for the necessary expensive device of high sensitivity chemical analysis.

Term " toxicant " especially comprises employed those poisonous organic solvent and additives in the art methods, ICH with its range 2 grades of solvents (ICH theme Q3C impurity: residual solvent), for example, dichloromethane.In addition, international guidelines (the internationalguidance for the development of therapeutic proteins) requirement that treatment is developed with albumen, in preparation process, should avoid harmful and noxious substance (see for details: international coordination meeting (InternationalConference on Harmonization) (ICH), theme Q3C; Www.emea.eu.int/).But advantageously, pastel of the present invention does not contain described 1 class toxicant.And the present invention only contains the solvent that ICH theme Q3C classifies as 3 classes, therefore, is fine acceptance in the treatment, and satisfies the requirement of administrative organization.

In addition, in a further preferred embodiment, biodegradable paste material of the present invention and moldable biomaterial do not contain infectious material.

Preferably, identical requirement for solvent also is effective for following substances usually: the plasticizer of biodegradable pastel, water-fast solid additive and/or water-soluble degraded regulator, and biodegradable pastel itself and moldable biomaterial of the present invention.

The variation of the concentration of biodegradable pastel of the present invention and moldable biomaterial causes being adapted to concrete medical application by the change of the mechanical property of the viscosity of pastel or moldable biomaterial, former position firm time, porosity and final implant.In addition, the variation of these parameters is the effective ways that adapt to the release dynamics of activating agent by the degradation behavior that changes water-fast polymer.

The D plasticizer

Term of the present invention " plasticizer " refers to organic liquid or solvent or its mixture pharmaceutically acceptable, water-soluble or that Yi Yushui mixes.The function of plasticizer is that dissolving is water-fast biodegradable, biocompatible and/or can biological resorbent polymer, so that water-soluble solid filling material suspends; Or dissolving makes the insoluble polymer of water-fast solid filler suspension.These functions depend on the character of activating agent.

Preferably, the function of plasticizer is to reduce water-fast glass transition temperature biodegradable, biocompatible and/or can biological resorbent polymer to be lower than the biomaterial moldable temperature that becomes, more preferably, water-fast glass transition temperature biodegradable, biocompatible and/or can biological resorbent polymer is lower than room temperature.

In the former position hardening process of preferred contact aqueous media or body fluid, plasticizer spreads stripping from pastel, stay aperture and cause forming stable compositions equipment or original position implant.Therefore, the glass transition temperature of polymer raises, and polymer solidifies and biomaterial is transformed into the implant of mechanically stable.In a preferred embodiment, described plasticizer is water-soluble or the solvent of Yi Yushui mixing.It can be liquid; Preferred plasticizer is water-soluble polymer.Preferably, plasticizer is lower on the impact of the glass transition temperature of the water-fast polymer in the implant of original position sclerosis, and compatible with activating agent.Depend on water-fast polymer, the plasticizer that is selected from the plasticizer that hereinafter further limits should use in the minimum mode of glass transition temperature impact on polymer after placement.

Term " dissolving " refers to substance dissolves or is suspended in the liquid, so that material is dispersed in the liquid.

Preferably, described plasticizer is biocompatible.More preferably, described plasticizer is selected from Polyethylene Glycol (PEG) 400, PEG200, PEG300, PEG600,1,3-butanediol, Oleum Ricini, C2 to C6 alkanol, propylene glycol, solketal, acetone, methyl acetate, ethyl acetate, ethyl lactate, methyl ethyl ketone, dimethyl formamide, dimethyl sulfoxine, dimethylsulfone, oxolane, decyl methyl sulfoxide, oleic acid, Allyl carbonate, N, N-diethyl-m-toluamide, 1-positive dodecyl aza cyclohepta alkane-2-ketone or its mixture.

Preferred biodegradable pastel of the present invention contains and is less than 60% plasticizer, more preferably less than 55%, and further more preferably less than 50%, most preferably 40% to 45%.

Term " biocompatible " refers to that material carries out the ability of suitable host response in concrete the application.And term " biocompatible " refers to that material does not show any toxicity, and can not cause any immunity or inflammatory reaction after application.

Term " biodegradable " specifically refers to such material, polymer for example, and the macromolecules degradation of its satellite internal diffusion and decomposing, but the evidence of not removing in the body exists.The minimizing of biodegradable quantity of material is the result of passive process in the body, and it is by the physical and chemical condition in the host tissue (for example, humidity, pH value) catalysis.

Term " can biological resorbent " specifically refers to such material, polymeric material for example, and it is through degraded and further absorb in vivo again; That is, polymer is because after the simple filtration or metabolism of degradation by-products, it is removed by natural way.Therefore, biology absorbs and refers to such concept, and it reflects whole eliminations of initial foreign material.In a preferred embodiment, described can biological resorbent polymer be a kind of like this polymer, and it is owing to the macromolecules degradation in the aqueous environments carries out chain interruption.Term " absorbs " again describes an active process.

The water-fast polymer of E

Term " water-fast polymer " refers to be not dissolved in the polymer in the water, namely when mixing with water, can not form homogeneous phase, it can be dissolved in the described plasticizer, and can in aqueous media, solidify to form the solid implant, therein, in case remove plasticizer, water-fast solid filler is injected towards surrounding tissue.Preferably, described water-fast polymer is " biocompatible ", " biodegradable " and/or " can be biological resorbent " polymer.More preferably, described water-fast polymer is a kind of aliphatic polymer, and the glass transition temperature of straight polymer is higher than 30 ℃.The intrinsic viscosity of polymer of the present invention (25 ℃, the viscosity that records in 0.1% chloroform) is about 0.1dl/g to 5dl/g, preferably about 0.1dl/g to 1dl/g.

In another embodiment, described polymer is the polymer that synthesizes.

Perhaps, described water-fast polymer is selected from polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polylactide Acetic acid, hydroxy-, bimol. cyclic ester (polyglactine), polyamide (PA), polymethyl methacrylate (PMMA), poly-methylol acrylic acid methyl ester. (PHEMA), polrvinyl chloride (PVC), polyvinyl alcohol (PVA), politef (PTFE) polyether-ether-ketone (PEEK), polysulfones (PSU), polyurethane, polysiloxanes or its mixture.

More preferably, described polymer is selected from poly-(alpha-hydroxy acid), poe, poly-anhydride, polyamino acid, polyglycolic acid (PGA), polylactic acid (PLLA), poly-(D, L)-lactic acid (PDLLA), poly-(D, the L-lactide-co-glycolide) or poly-(L-lactide-co-glycolide) (PLGA), poly-(lactic acid-altogether-hydroxyacetic acid) Polyethylene Glycol (PLGA-PEG) copolymer, poly-(3-hydroxybutyrate) (P (3-HB)), poly-(3-hydroxypentanoic acid) (P (3-HV)), poly-to dioxanone (PDS), gather (6-caprolactone) (PCL), poly-anhydride (PA), its copolymer, terpolymer, block copolymer, compositions or mixture.

In another embodiment of the invention, water-fast polymer is terminated polymer.Term " terminated polymer " refers to that the free carboxyl group group of linear polymer chain is by pure esterification.

In another embodiment of the invention, water-fast polymer is the PLGA-PEG copolymer, the two blocks of preferred PLGA-PEG or triblock copolymer.

The water-fast solid filler of F

Term " water-fast solid filler " refers to water insoluble and chemical compound plasticizer,, does not form homogeneous phase when mixing with water or plasticizer that is.

In case moldable biomaterial sclerosis, described water-fast solid filler is as the substrate in the biodegradable paste material.And described water-fast solid filler can further increase biocompatibility (for example, cell adhesion), with the pH value of stabilizing local in polymer degradation processes.

Preferably, described water-fast solid filler is inorganic or organic compound.

Term " calcium phosphate " comprises the chemical compound that contains following ion: calcium ion (Ca ²⁺), phosphate anion (PO ₃ ^3-), randomly, other ion is such as hydroxide ion (OH ^-), carbonate example (CO ₃ ^2-) or magnesium ion (Mg ²⁺) or be suitable for other ion of the water-fast solid-filling agent of the present invention.

The degraded regulator that G is water-soluble

Term " water-soluble degraded regulator " refer to pharmaceutically acceptable and in the waterborne liquid such as water or body fluid inflatable or the dissolving chemical compound, when being added into biodegradable paste material, it can increase the porosity of the moldable biomaterial in external or the body.For example, the amount of used water-soluble degraded regulator is depended in the increase of the porosity of the solid implant of formation.Preferably, water-soluble degraded regulator increases the hole, the quantity of the macropore of preferred sufficient size, and described hole is used for entering the living cells of former position hardened material in interior growth.More preferably, water-soluble degraded regulator allows the degraded of the polymers compositions of the biodegradable paste material of adjusting.

On the other hand, the degraded regulator can damaged place fix or enrichment in give birth to somatomedin, further promote regenerative process, such as but not limited to bone lengthening.In addition, degraded regulator (for example, sweller) can form the hydrogel in the moldable biomaterial when contacting with water, and it is similar to the character of the blood clot of self-assembling formation.

Water-soluble degraded regulator of the present invention comprises, for example, sodium alginate, amylase, amylopectin, starch, hyaluronic acid, hyaluronate sodium, colloid, collagen, carboxymethyl cellulose, methylcellulose, carboxymethylcellulose calcium salt, hydroxypropyl emthylcellulose, hydroxy butyl methyl cellulose, hydroxyethyl-cellulose, or methyl hydroxyethylcellulose, and derivant.

In another embodiment, water-soluble degraded regulator is surfactant, and the block copolymer of optimization ethylene oxide/sorbitan and expoxy propane is such as Pluronics Or Tween

80 (for example, polysorbate80, Montanox

80, polyoxyethylene sorbitol acid anhydride oleate).

More preferably, water-soluble degraded regulator is carboxymethyl cellulose salt, sanlose most preferably, and randomly, particle diameter is less than 1000 μ m, and more preferably particle diameter is 25-1000 μ m.Preferably, the percentage by weight of sanlose is less than 10wt%, preferably less than 5wt%, and more preferably 1-4wt%, 2-3.5wt% most preferably is based on the gross weight of biomaterial pastel component.

Term of the present invention " particle diameter " refer to micron order (μ m) such as being evenly distributed of the material diameter of tricalcium phosphate or carboxymethyl cellulose, can measure by screen analysis or laser diffraction.The concrete particle size range of material for example can obtain by screening.

The H activating agent

Term " activating agent " comprises polypeptide or small-molecule drug.

Be appreciated that activating agent preferably can not assembled because of the precipitation after implanting or microprecipitation and part or all of inactivation.For example, this can realize by evenly applying on the solid particle porous material, and is described such as WO03/043673.

Term " evenly coating " or " evenly distributing " refer to that activating agent is distributed evenly at inner surface and/or the outer surface of granular solids porous material.

Be evenly distributed with and be beneficial to activating agent effectively release and the active implantation position tissue on every side that enters of maintenance.And, be appreciated that activating agent can not assembled because of precipitation or microprecipitation and part or all of inactivation, realized the adhering to of albumen of bioactive, non-gathering by even coating.

Term " bone conduction " refers to be provided for the angioaccess cellular infiltration and absorption, cartilage form and the material of the good porous support of cartilaginous tissue deposition.The bone conduction material can be by the generation of support effect support rib.

Term " bone is induced " refers to the cell mesenchymal stem cells MSCs is changed into the ability of osteoblast and chondrocyte.The prerequisite that bone is induced is a signal, and it is distributed in the surrounding tissue by moldable biomaterial, around foregoing osteoblast precursor activation in the tissue.Bone used herein is induced and is comprised that Interstitial cell is divided into bone precursor, osteoblast.And bone is induced and is comprised that also described osteoblast is divided into osteocyte, ripe osteocyte.And bone is induced and is comprised that also Interstitial cell is divided into chondrocyte.Especially in long bone, the chondroblast and the chondrocyte that are present in the perichondrium of bone also can be divided into osteocyte.Therefore, bone induces the cell differentiation of differentiation that requirement is not broken up or less for forming the osteocyte of bone.Therefore, the prerequisite that bone is induced is a signal, and it distributes by moldable biomaterial and enters surrounding tissue, usually has foregoing osteocyte precursor in the tissue around.

Term " bone formation " is described by the synthetic new bone of osteoblast.According to the present invention, the osteocyte that is pre-existing in around transplanting place or the moldable biomaterial or CFU-GM form the moldable biomaterial of the sclerosis of the moldable biomaterial structure that adopts sclerosis, especially in hardening process, form, can depend on substrate thereon as cell (for example, osteocyte).

The protein and the peptide that are included in the moldable biomaterial of the present invention preferably have body internal skeleton inducing properties.For example, transforming growth factor-beta well known in the art (TGF-β) superfamily comprises the member with bone inducing properties.The member of described TGF-beta superfamily lists hereinafter.In a word, by the osteoinductive protein matter of the moldable biomaterial of the present invention after discharging from carrier or the peptide bone inducement signal as the bone precursor of the implant site surrounding tissue of moldable biomaterial.

Term " bone is induced polypeptide " refers to polypeptide, the member of transforming growth factor-beta (TGF-β) superfamily for example, and it has the bone inducing properties.

In another preferred embodiment of the moldable biomaterial of the present invention, described osteoinductive protein matter is the member of TGF-β family.

The GDF of TGF-β family has shown and has related to the regulate several biological processes that comprises bone formation.All members of described family are the secrete polypeptides that contain the feature domain structure.Terminal at N-, the TGF-β member of family comprises signal peptide or secretion tendon (secretion leader)., this sequence is connected on the C-end by predomain (prodomain) and mature polypeptide sequence.The mature polypeptide sequence comprises seven conservative cysteine, wherein six formation that are used for intramolecular disulfide bond, a dimerization that is used for two polypeptide.The biological activity TGF-β member of family is dimer, preferably is comprised of two mature polypeptides.The TGF-β member of family secretion is the protein that comprises the predomain except mature sequence.Described predomain divides in the extracellular and is not the part of signaling molecule.

In the present invention, the protein of term " the TGF-β member of family " or the described family mentioned below comprises described protein or member's all biological activity variants and all variants and their nonactive precursor.Therefore, only comprise the protein of mature sequence and comprise mature sequence and the protein of predomain or mature protein, predomain and targeting sequencing, and bioactive fragment or its variant are all within the scope of the invention.Whether the TGF-β member of family fragment has biological activated energy is easily measured by the biological detecting method of description of the Prior Art.

More preferably, the TGF-beta superfamily member is the member of BMP or GDF subtribe.

Bone of the present invention induces polypeptide to be preferably selected from BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14, BMP-15, BMP-16, GDF-1, GDF-2, GDF-3, GDF-4, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10 and GDF-11.Most preferably, described bone induces polypeptide to be selected from BMP-2, BMP-7 and GDF-5.

The disclosed bone of publication induces polypeptide to comprise: OP-1 and OP-2: U.S. Patent No. 5,011, and 691, U.S. Patent No. 5,266,683, the people such as Ozkaynak, (1990) EMBO is J.9:2085-2093; OP-3:WO94/10203 (PCT US93/10520); BMP2, BMP3, BMP4:WO88/00205, the people such as Wozney, (1988) Science 242:1528-1534; The people such as BMP5 and BMP6:Celeste, (1991) PNAS 87:9843-9847; The people such as Vgr-1:Lyons, (1989) PNAS 86:4554-4558; The people such as DPP:Padgett, (1987) Nature 325:81-84; Vg-1:Weeks (1987) Cell 51:861-867; BMP-9:WO95/33830 (PCT/US95/07084); BMP-10:WO94/26893 (PCT/US94/05290); BMP-11:WO94/26892 (PCT/US94/05288); BMP-12:WO95/16035 (PCT/US94/14030); BMP-13:WO95/16035 (PCT/US94/14030); People (1991) the PNAS 88:4250-4254 such as GDF-1:WO92/00382 (PCT/US91/04096) and Lee; GDF-8:WO94/21681 (PCT/US94/03019); GDF-9:WO94/15966 (PCT/US94/00685); GDF-10:WO95/10539 (PCT/US94/11440); GDF-11:WO96/01845 (PCT/US95/08543); BMP-15:WO96/36710 (PCT/US96/06540); MP121:WO96/01316 (PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO94/15949 (PCT/US94/00657) and WO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350); GDF-6 (CDMP-2, BMP13): WO95/01801 (PCT/US94/07762) and WO96/14335 and WO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3, BMP-12): WO95/10802 (PCT/US94/07799) and WO95/10635 (PCT/US94/14030).

Preferably, the activating agent of BMP or GDF subtribe, for example BMP-2, BMP-7 or GDF-5 refer to respectively pre-precursor (preproform), precursor (proform) or ripe (for example, BMP-2-, BMP-7-or GDF-5-) peptide.Comprise in addition having essentially identical biological activity, fragment and the variant of the described protein of preferred bone inducing properties.

Be included in the variant that also has described protein within the present invention, for example has essentially identical bioactive BMP-2 variant, it comprises the clipped form of the polypeptide that for example ripe BMP-2 protein sequence and preamble mention, described ripe BMP-2 protein sequence comprises that the N-end such as the alanine extension of N-end extends, such as people such as Ruppert, described in (1996) Eur.J.Biochem., 237:295-302.

Preferably, described activating agent is not glycosylated protein, more preferably is derived from the recombiant protein of E.coli.The advantage of glycosylated protein is not, for example, prolongs fixing at damaged place and/or has reduced requirement such as the activating agent of rhBMP-2.

The present invention also comprises such embodiment, and wherein said activating agent is selected from hormone, cytokine, somatomedin, antibiotic and other natural and/or synthetic medicine, such as steroid, prostaglandin etc.

Preferably, described activating agent is parathyroid hormone (PTH) and/or PTH1-34 peptide.

In another embodiment of the invention, described activating agent is " chondrocyte induction " or " regenerating bone or cartilage " protein.Preferably, chondrocyte induction protein is MIA/CD-RAP (MIA, melanoma suppresses active, cartilage derived tretinoin sensitive Protein, EP0710248, EP1146897), OTOR (is derived from fibrocellular protein, FDP, class MIA (MIA-like), MIAL) and TANGO130 (people such as Bosserhoff, (2004), Gene Expr.Patterns.4:473-479; Bosserhoff and Buettner (2003), Biomaterials 24:3229-3234; The people such as Bosserhoff (1997), Dev.Dyn.208:516-525; WO00/12762), human MIA/CD-RAP more preferably.

The I implant

Described " implant " refers to armarium, orthopedic device or biomaterial.Preferably, described implant is spinal implant, fracture repair implant, be used for bone defect, critical size is damaged and the implant of nonunion, be used for repair of cartilage, maxillary surface reproduces and the implant of joint reconstruction, paradental defect implant, the implant used as implant or other plastic surgery operations of bone cavity implant, such as cage, plate, screw, pin, fixing device.

Term " spinal implant " has a detailed description hereinbefore.

Description to figure

Describe each aspect of the present invention in detail in conjunction with Fig. 1-5.

Fig. 1 shows the inside and outside porosity of the moldable biomaterial of the present invention's two components after the sclerosis of aqueous environments situ.Compositions shown in the figure is as follows: bata-tricalcium phosphate granule (40.0wt%), polymer pasty state thing (60.0wt%), described polymer pasty state thing comprise the ratio of lactic acid/hydroxyacetic acid be 50: 50 and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (22.2wt%), PEG400 (44.4wt%), bata-tricalcium phosphate powder (33.3wt%).

Image A shows the outer surface of the moldable biomaterial of the present invention's two components after the original position sclerosis, shows porosely, and described hole is especially based on the hole between the bata-tricalcium phosphate granule.

Image B shows the inside of material, shows diameter greater than the hole of 100 μ m, and it is the around basic demand of the globality of in-house implant material.

The advantage of the moldable biomaterial of the present invention's two components is that it has moldable bonding viscosity, is easy to be suitable for application site and is retained in application site.With other biodegradable implant material, such as bata-tricalcium phosphate granule or HA nanoparticle suspension phase ratio, implant of the present invention is being used for moistening open environment, surgical field for example, and during such as the severe haemorrhage environment, it has the well anti-ability that washes out.In addition, embedded material can be easily be used for being filled into the implant such as the various spine cages that occur on the market, and can not leak material and washed out.In addition, described material has the characteristic of bonding support after implantation, and it stands the mechanical pressure of surrounding tissue.Another advantage of comparing with other Injectable biomaterials is the loose structure of implant material after the sclerosis of former position in vivo or in the tissue, and with the compression resistance of comparing such as collagen base implant.

The shortcoming of biomaterial that forms the larger integral body of porous matrix is because their hardness, its can not with the implant of hollow, use together (bottleneck) such as spine cage.Because moldable viscosity and easily application, implant of the present invention is conducive to as the bone graft substitute biomaterial, to fill spinal implants, such as the spine cage of various shapes, it forms the overall structure of implant material in cage after the sclerosis of former position.

Fig. 2 shows the other external pores rate of the moldable biomaterial of the present invention's two components after the original position sclerosis, and described other external pores degree is by adding the carboxymethyl cellulose acquisition of expanding in the pastel component.Used compositions is as follows: bata-tricalcium phosphate granule (40.0wt%), polymer pasty state thing (60.0wt%), described polymer pasty state thing comprise the ratio of lactic acid/hydroxyacetic acid be 50: 50 and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (21.6wt%), PEG400 (43.1wt%), bata-tricalcium phosphate powder (32.3wt%) and sanlose (3.0wt%).

Image A shows the outer surface of the moldable biomaterial of the present invention's two components, has shown the extra hole of comparing with the implant material of Fig. 1, and described hole is formed by the expansion of sanlose.These holes have the diameter greater than 100 μ m, have satisfied the basic demand of cell in interior growth.

The advantage that adds sweller such as sanlose is, increases the porosity of the outer surface of implant material, and wood interior porosity (image B) can not increase when adding sweller.Wood interior porosity is by such as the formation of the grain bed of the solid filler of bata-tricalcium phosphate and go out degradable paste material by solvent exchange and set up.

Fig. 3 shows the contrast of the mechanical stability of the mechanical stability of the 2 hours post polymerization thing pastel that harden in position and the moldable biomaterial of the present invention's two components.White post representative is according to the polymer pasty state thing of embodiment 2 preparations, and it has following compositions: the ratio of lactic acid/hydroxyacetic acid be 50: 50 (RG502H) and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (21.6wt%), PEG400 (43.1wt%), bata-tricalcium phosphate powder (32.3wt%) and sanlose (3.0wt%).The representative of black post is according to the implant material of embodiment 3 preparations, and it has following compositions: according to the bata-tricalcium phosphate granule (40.0wt%) of embodiment 1 preparation and polymer (biodegradable) pastel (60.0wt%) of describing in conjunction with white post according to embodiment 3.

An aspect of of the present present invention is, although with polymer pasty state phase ratio, the polymer content of implant material has reduced, but implant material has shown the hardness after 2 hours surprisingly, and it is higher 2.5 times than the polymer pasty state thing hardness that does not add porous ceramic film material.

Fig. 4 shows the organic solvent that depends on for the preparation of biodegradable pastel, i.e. the components b of the moldable biomaterial of the present invention) protein stability.Pastel shown in Figure 4 is by following embodiment 6 described method preparations.A represents control sample, and B is PEG400, and C is N-Methyl pyrrolidone, and D is dimethyl sulfoxide, and E is glycofurol.

This figure has given prominence to (part) degraded that can impel the latter that contacts between organic solvent and the protein.As shown in the figure, degradation rate (white post) can reach 75% of used protein primary quantity after 48 hours.

An advantage of the present invention is, organic solvent is to the negative effect on the active substance that contains in the implant material, polymer pasty state thing that can be by will containing organic solvent between the storage life and contain to separate such as the active substance of the ceramic material of bata-tricalcium phosphate granule and be eliminated.Before implant material is applied to the patient, mix immediately with the pastel that contains organic solvent by containing ceramic activity of materials material, with protein and comparing that the substrate that contains organic solvent is combined, the activity of active substance such as bone growth inducing protein can be saved.

Fig. 5 represents the variation of the degree of hydrolysis of the polymer that the pastel component of the moldable biomaterial of the present invention is interior.During degree of hydrolysis passes through and the quantitative determination of the required sodium hydroxide solution of the acid degradation product of pastel component of the moldable biomaterial of 1 gram.In Fig. 5, the PGLA copolymer is as the polymers compositions (seeing embodiment 7) of biodegradable paste material.

Wherein the Lycoperdon polymorphum Vitt triangle represents the pastel component of moldable biomaterial, by the ratio of lactic acid/hydroxyacetic acid be 50: 50 and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (33.3wt%) and PEG400 (66.6wt%) form; White square represents the pastel component of the moldable biomaterial of the present invention, by the ratio of lactic acid/hydroxyacetic acid be 50: 50 and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (22.2wt%), PEG400 (44.5wt%) and bata-tricalcium phosphate powder (33.3wt%) form; Black square represents the pastel component of the moldable biomaterial of the present invention, by the ratio of lactic acid/hydroxyacetic acid be 50: 50 and molecular weight be 13.6kDa poly-(lactic acid-altogether-hydroxyacetic acid) (21.6wt%), PEG400 (43.1wt%), bata-tricalcium phosphate powder (32.3wt%) and sanlose (3.0wt%) form.

The titration curve of three samples shows that the degraded of polymer (referring to the PLGA copolymer herein) has been accelerated in the interpolation of water-fast inorganic filler (referring to bata-tricalcium phosphate herein) surprisingly.

In addition, the high concentration of water-soluble degraded regulator such as used about 3% carboxymethyl cellulose among Fig. 5, has been accelerated the degraded of the polymer in the implant material, thereby has been changed the release profiles of active component.

An advantage of the present invention is, the pastel component, and namely biodegradable paste material and granular solids porous material such as graininess phosphoric acid calcium mineral, form composite interstitial substance, and it is particularly advantageous in bone and replaces or bone lengthening.Described substrate keeps its structure (physics) integrity at least in time of two to three days after implantation, and keep the loose structure of calcium phosphate granules within several weeks in the biotic environment that bone replaces occurs.Structure (physics) integrity refers to shape and the size of the substrate that basic maintenance is implanted.This with after implantation immediately or the compositions that collapses in the short time without the non-porous substance of determining form compare.The advantage of described substrate is to keep its porosity, and this is very important for bone replacement or bone lengthening process.

Because two stage degraded, implant material of the present invention have kept being used for improving osteoplastic loose structure.In addition, two stage degraded makes active substance such as bone growth inducing agent controlled release or is sent to surrounding tissue.The release that the phase I degraded of the polymer in the pastel component of the moldable biomaterial of the present invention of former position sclerosis causes can change with the variation of water-fast solid filler and/or water-soluble degraded regulator.

Fig. 6 shows the recovery of the rhBMP-2 that is bound by various biomaterials.

As shown in Figure 6, the sample demonstration that only contains β-TCP granule almost interacts with rhBMP-2 (E.coli) nothing, namely almost 100% reclaims rhBMP-2 from the supernatant (A).Because rhBMP-2 is positively charged, thereby the electronegative group in the moldable biomaterial of the present invention can cause the minimizing of reclaiming.

It shows that the polymer of non-end-blocking and CMC are fit to mode for triggering and/or promoting the rhBMP-2 activating agent to be adsorbed to the moldable biomaterial of the present invention.The absorption of improved activating agent is relevant from the prolongation of the moldable biomaterial sustained release of the present invention activating agent when using in vivo.

Because the absolute magnitude of the β of each sample-TCP granule equates, and the sample demonstration that only contains β-TCP granule almost interacts with rhBMP-2 (E.coli) nothing, the carboxyl anion triggering that the absorption of the protein that is adsorbed to other carrier of observing must be introduced by PLGA copolymer and CMC by non-end-blocking respectively.

In fact, experiment shows that the absorption (B to D) of the protein that is adsorbed to other carrier of observing is triggered by the carboxyl anion that PLGA copolymer and CMC by non-end-blocking introduce respectively.This conclusion, the prescription (D) that namely contains the PLGA copolymer of end-blocking produces the response rate of rising than the prescription (B and C) of the PLGA copolymer that contains non-end-blocking, has obtained the support of observed result.

Fig. 7 shows the degraded of the polymer of the two kinds of different biomaterials of passing in time.A representative is according to the degraded of the biodegradable paste material that is comprised of following substances of embodiment 2 preparations: dead plaster (20.6wt%) and the sanlose (1.0wt%) of Resomer RG504 (44.0wt%), PEG 400 (22.0wt%), Biocement D (20.6wt%), drying.B shows the degraded of the moldable biomaterial of embodiment 8.

Data show is compared with B, and the degraded that the polymer in the biomaterial of A is passed has in time prolonged, thereby causes material B again absorption early.Data have also shown the three stage degradation kineticss (see Fig. 7 B, decline stage 0-1 days, 2-4 days and 7-10 days) of the moldable biomaterial of the present invention typically.

Embodiment

Embodiment 1: preparation has applied the solid particle porous material of activating agent

Present embodiment use applied β-TCP granule as solid porous material and rhGDF-5 as activating agent.Replacement scheme can prepare similarly.

Raw material must be sterilized in a suitable manner.At first 500mg β-TCP (500-1000 μ m granular size) is positioned in the 2R-glass with dried forms.RhGDF-5 stock solution (3.4mg/ml is in 10mM HCl) is diluted to 0.54 μ g/ml with corresponding coated buffer.It is upper and be absorbed to β-TCP that the rhGDF-5 solution of the 475 μ l that will be obtained by said method with suction pipe moves liquid.Moistening granular substance is in 25 ℃ of lower cultivations 1 hour, then lyophilizing.Other embodiment of coating β-TCP has description in WO 03/043673 and PCT/EP2005/006204.

Embodiment 2: prepare biodegradable paste material

Initial polymer (RG502H; PLGA; Polymer composition: 48-52mol%D, L-lactide and 48-51mol% Acetic acid, hydroxy-, bimol. cyclic ester; Intrinsic viscosity: 0.16-0.24dl/g, 25 ℃, 0.1% in CHCl ₃In; Boehringer, Ingelheim) be added in the organic solvent (PEG400) of the necessary amounts in the porcelain crucible.These two kinds of components homogenize, and heat until polymer is dissolved in the organic solvent fully at about 60 ℃.Subsequently, inorganic filler (bata-tricalcium phosphate powder) and optional other excipient (for example, the degraded regulator is such as sanlose) are dispersed in the polymer solution.

Embodiment 3: the moldable biomaterial that comprises the former position sclerosis of porous calcic pottery

The coating of embodiment 1 the bata-tricalcium phosphate granule and the biodegradable paste material of embodiment 2 in crucible with the mix homogeneously mildly of sterile spoon for example, to form the moldable material of thickness.The different implant material of ratio (wt%/wt%) of preparation bata-tricalcium phosphate granule and polymer pasty state thing: a) β-TCP: the ratio of polymer pasty state thing is 1: 1.3; B) β-TCP: the ratio of polymer pasty state thing is 1: 1.4; C) β-TCP: the ratio of polymer pasty state thing is 1: 1.5; And d) β-TCP: the ratio of polymer pasty state thing is 1: 1.7.

For requiring biodegradable paste material or moldable biomaterial to be in all experiments of the rear shape of its sclerosis, described material transfer is (every hole 250-300mg) to the hole of 48-orifice plate.Then described orifice plate is cultivated in the bath that contains the PBS-buffer, and wherein temperature is fixed on 37 ℃.Described bath is with 150min ^-1Frequency constantly vibrate.

Embodiment 4: mechanical test

Biodegradable paste material (implant material) by the former position sclerosis of the moistening sample of the hardening of the biodegradable paste material of embodiment 2 preparation and embodiment 3 preparations, be transferred in the hole of 96 orifice plates (every hole 150-200mg, three holes of each time point and sample).The described orifice plate that contains subsequently sample is transferred to be cultivated in the bath, and the temperature of bath remains at 37 ℃, and with the simulation physiological condition, wherein the PBS-buffer is as cultivating medium.In the moment of every appointment, 96 orifice plates shift out from cultivate bath, to carry out mechanical test.

The hardness of sample adopts TH2730 (Fa Thuemler) test.This machine is mainly by the Metal-Piercing instrument that can exert pressure at sample be used for the power that control and measurement applies and determine to form at the LVDT-sensor of the capped distance of measuring process.Before the different samples of test, need definite height (h that does not contain the hole of any sample ₁).Therefore, the start-up point that is used for the punching tool of following measurement is fixed.The practical measurement of sample hardness comprises two steps.In measuring, the first step must determine the height (h of concrete sample ₂), wherein the crosshead speed (crossheadvelocity) of punching tool is per minute 40mm, the power that applies is restricted to 0.2N.Carrying out the second step measurement is the distance (d) that punching tool is capped in sample in 30 seconds time of measurement, and the power that wherein applies remains 20N always.The hardness calculation of sample is as follows:

Hardness (%)=(h ₂-d)/h ₂* 100%

Described method is based on the hardness measurement according to Shore (DIN53505).

Embodiment 5: analyze for the preparation of SEM-

According to standardization program well known by persons skilled in the art, the golden sputter of sample hardening and vacuum drying.Use the voltage of 20kV and finish the SEM-microphotograph.The object construction that is used for these analyses is the surface of graininess sample of implant material and core and the porosity that especially shows by these structures.

Embodiment 6: the stability of the rhGDF-5 in the different organic solvents

The solvent of use such as PEG400, N-Methyl pyrrolidone, dimethyl sulfoxide and glycofurol.By preparing sample and object of reference at 500mg β-TCP coating rhGDF-5, to obtain ultimate density as β-TCP of 500 μ g/g.Subsequently, 666 μ l solvent separately is added in every duplicate samples, and object of reference does not process.After 24 hours, sample and object of reference all adopt the 3ml Extraction buffer to extract 1 hour at 4 ℃ 25 ℃ of lower cultivations, and described buffer is comprised of carbamide (8M), Tris (10mM) and EDTA (100mM), and its pH value is adjusted to 6.7 with hydrochloric acid.Behind extraction step, all samples and object of reference were with 4500rpm centrifugalize 3 minutes.Subsequently, the supernatant is diluted with the solvent orange 2 A (0.15% trifluoroacetic acid and 20% acetonitrile are in water) of 1: 1 ratio.Solvent B is 0.15% trifluoroacetic acid and 84% acetonitrile in water.Adopt Vydac C18, specification is 2.1 * 250mm, and flow velocity is 0.3ml/min, measures the characteristic of protein.By measuring the absorptance record elution profile at 220nm place.The amount of rhGDF-5, rhBMP-2 and their catabolite is by the calculated by peak area at 220nm place.

Embodiment 7: measure the degraded of polymer

The biodegradable paste material of embodiment 2 preparations is accurately weighed in 6R-Vail, adds therein about 3mlPBS-buffer.For the pH value of show sample, in sample, add 20 μ l bromthymol blues, wherein navy blue indication pH neutral.The degraded of polymer (referring to the PLGA copolymer herein) causes the reduction of pH value, and this is shown by the change color from the navy blue to the yellow.To the time point of appointment, the titration of 0.04M sodium hydroxide solution is adopted in the supernatant of sample, until the pH value of sample reaches neutral, this is by the navy blue indication of indicator.To each time point, the summation of the total amount of the sodium hydroxide of consumption, and the standardization by the use amount of considering the PLGA copolymer.

Embodiment 8: the content of polymer in the polymerization in vitro thing degraded of in time passage of mensuration and the moldable biomaterial

With 10.0g β-TCP granule and the biodegradable paste material of 15.0g (ResomerRG502H) (22.2wt%), PEG400 (44.5wt%), β-TCP powder (20.8wt%) and dry calcium sulphate dihydrate (12.5wt%) mix.Get the resulting thick substances of 1.0g to form cylindrical sample, it is transferred in the 50ml polypropylene reaction tube that the 50ml physiological phosphate buffer is housed subsequently.

At the time point (after cultivating 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days) of appointment, sample is removed and vacuum drying.The vacuum drying composition material of the about 75mg of accurate weighing in 1.5ml polypropylene reaction tube.Subsequently, add the 1.0ml oxolane.Stir (300min in lasting level ^-1) under in room temperature with sample culturing 10 minutes.By insoluble inorganic constituents being separated from polymer solution in 5 minutes with the 13000rpm centrifugalize.Then, the supernatant that obtains is analyzed through assembled dimension exclusion chromatography multi-angle light scattering apparatus, and described instrument is especially by HPLC equipment, size exclusion post (7.8mm * 30.0cm) and forming in conjunction with the multi-angle scatter detector of refractive index detector in turn.

For measuring the molecular weight of the polymer that from sample separately, extracts, inject the 200 μ l supernatantes.Adopt oxolane with constant flow rate 1.0ml/min eluting polymer.Column temperature increases to 40 ℃.In order to make used software can calculate the absolute injection rate of absolute molecular weight and the polymer of analyzing, the different differential refractive index (dn/dc) of each polymer are measured in advance by the area under the refractive index signal curve that records each polymer concentration.By continuing similarly to process organic plasticizer, this method allows to measure the in time related component of the moldable biomaterial of passage.

The interaction of embodiment 9:rhBMP-2 (E.coli) and the various components of moldable biomaterial

75mg β-TCP granule mixes with the biodegradable paste material of the 112.5mg for preparing according to above-described embodiment, to obtain moldable biomaterial.Use subsequently the variant of following various biomaterials:

A) β-TCP granule

B) β-TCP granule+biodegradable paste material, described biodegradable paste material is by PEG400 (44.5wt%), β-TCP powder (33.3wt%) and Resomer

RG502H (non-end-blocking, 22.2wt% purchases the Ingelheim to Boehringer)

C) β-TCP granule+biodegradable paste material, described biodegradable paste material is by PEG400 (43.0wt%), β-TCP powder (32.4wt%), Resomer RG502H (non-end-blocking, 21.6wt% purchases the Ingelheim to Boehringer) and DS be 0.7 and particle diameter be 100-200 μ m sanlose (CMC) (3.0wt%)

D) β-TCP granule+biodegradable paste material, described biodegradable paste material is by PEG400 (44.5wt%), β-TCP powder (33.3wt%) and Resomer RG502 (end-blocking, 22.2wt% purchases the Ingelheim to Boehringer)

For distinguish biodegradable paste material on the distribution of the impact of the interactional degree of rhBMP-2 (E.coli) and the β-TCP granule difference to whole protein adsorption, adopt 75mg β-TCP as reference carrier (A).

Every duplicate samples is transferred in the 15ml polypropylene reaction tube, be filled with in the described reaction tube 15ml aqueous buffer solution (60mM calcium chloride, 0.01wt% polysorbate80,0.02wt% Hydrazoic acid,sodium salt in 20mM morpholino b acid monohydrate (MES) solution, pH6.2).All samples is with 30 μ grhBMP-2 (E.coli) mark-ons.At the time point (1 day, 2 days, 4 days, 7 days, 10 days) of appointment, use 250mm * 4.6mm C4 post (Vydac) to measure rhBMP-2 (E.coli) concentration in each sample supernatant by RP-HPLC.

The 20wt% acetonitrile of Yu Shuizhong and the 84wt% acetonitrile of 0.15wt% trifluoroacetic acid and Yu Shuizhong and 0.15wt% trifluoroacetic acid are respectively as eluent.Flow velocity increases to 0.8ml/min.Measure concentration by the fluoroscopic examination that 340nm (exciting 280nm) locates.The amount (100% reclaims) of rhBMP-2 in the supernatant of locating for " zero " with respect to time point, the amount of the rhBMP-2 in the mensuration supernatant.

Claims

1. a moldable biomaterial comprises

A) a kind of granular solids porous material, its particle diameter are 100-4000 μ m, and

B) a kind of biodegradable paste material,

Wherein

A) described granular solids porous material comprises the ceramic granule, and described ceramic granule is that the tricalcium phosphate of 100-4000 μ m is made by mean diameter; And

B) described biodegradable paste material is a kind of pastel, and it comprises

I. plasticizer, it is the biocompatible organic liquid that a kind of water-soluble or Yi Yushui mixes, described plasticizer is selected from by PEG400, PEG200, PEG300, PEG600,1, the 3-butanediol, Oleum Ricini, METHYLPYRROLIDONE, 2-Pyrrolidone, C2 to C6 alkanol, propylene glycol, solketal, acetone, methyl acetate, ethyl acetate, ethyl lactate, methyl ethyl ketone, dimethyl formamide, dimethyl sulfoxine, dimethylsulfone, oxolane, decyl methyl sulfoxide, oleic acid, Allyl carbonate, N, N-diethyl-m-toluamide, the group of 1-positive dodecyl aza cyclohepta alkane-2-ketone or its compositions of mixtures;

Ii. water-fast polymer, it is dissolved in described plasticizer and is biocompatible, biodegradable and/or can be biological resorbent, described water-fast polymer is selected from by poly-(alpha-hydroxy acid), poe, poly-anhydride, polyamino acid, polyglycolic acid, polylactic acid, poly-(lactic acid-altogether-hydroxyacetic acid), poly-(lactic acid-altogether-hydroxyacetic acid) ethylene glycol copolymer, poly-(3-hydroxybutyrate), poly-(3-hydroxypentanoic acid), poly-to dioxanone, poly-(6-caprolactone), poly-anhydride poe, or its copolymer, the group of compositions and/or compositions of mixtures; And

Iii. water-fast solid filler, it is insoluble to described plasticizer, and

Iiia) be selected from calcium compounds, magnesium oxide, magnesium hydroxide, magnesium carbonate, silicon dioxide or its compositions or mixture; Perhaps

Iiib) be selected from chitosan, collagen, calcium alginate, poly-(2-hydroxyethyl methylacrylate), hyaluronic acid, cellulose or starch.

2. moldable biomaterial according to claim 1 is characterized in that, described polylactic acid is poly-(D, L)-lactic acid, and described copolymer is selected from terpolymer and block copolymer.

3. moldable biomaterial according to claim 1, it has moldable viscosity, and in case contact with aqueous media, can harden to form the solid implant at former position.

4. moldable biomaterial according to claim 1, wherein said component a) and b) use with the ratio that can form bonding product.

5. moldable biomaterial according to claim 1, wherein components b) pastel comprise water-soluble degraded regulator, it is carboxymethyl cellulose.

6. moldable biomaterial according to claim 1 further comprises

C) a kind of activating agent.

7. moldable biomaterial according to claim 6, wherein said activating agent is skeletal growth factor.

8. moldable biomaterial according to claim 6, wherein said activating agent is selected from the group that is comprised of BMP-2, BMP-7 and GDF-5.

9. moldable biomaterial according to claim 1, it is presented at the two stage degraded at former position.

10. moldable biomaterial according to claim 1 keeps physical integrity in 2-3 days at least time after it hardens in position, and keeps porous prilled structure after the polymers compositions degraded.

11. an independent component that comprises each described moldable biomaterial among the claim 1-10 a) and b) or claim 6-8 in each described moldable biomaterial isolated component a), b) and kit c).

12. a component that comprises each described moldable biomaterial among the claim 1-10 a) and b) or claim 6-8 in each described moldable biomaterial component a), b) and implant c).

13. method for preparing moldable biomaterial, it is that granular porous calcium phosphate or the calcium sulfate of 100-4000 μ m mixes that described method comprises pastel and mean diameter, so that the gained mixture has moldable viscosity, it is in case contact and can harden at former position with aqueous media, to form solid porous implant, wherein said pastel comprises

Iii. water-fast solid filler, it is insoluble to described plasticizer, and

14. method according to claim 13 is characterized in that, described polylactic acid is poly-(D, L)-lactic acid, and described copolymer is selected from terpolymer and block copolymer.

15. the application of each described moldable biomaterial among the claim 1-10, for the preparation of pharmaceutical composition or armarium, spinal fusion, bone defect, critical size are damaged to be used for, nonunion, joint relocate, fracture repair, repair of cartilage, maxillary surface are reproduced, periodontal reparation, lumbar intervertebral disc degeneration disease, spondylolisthesis disease, osseous tissue are filled.

16. the application of the described kit of claim 11, for the preparation of pharmaceutical composition or armarium, spinal fusion, bone defect, critical size are damaged to be used for, nonunion, joint relocate, fracture repair, repair of cartilage, maxillary surface are reproduced, periodontal reparation, lumbar intervertebral disc degeneration disease, spondylolisthesis disease, osseous tissue are filled.

17. the application of the described implant of claim 12, for the preparation of pharmaceutical composition or armarium, spinal fusion, bone defect, critical size are damaged to be used for, nonunion, joint relocate, fracture repair, repair of cartilage, maxillary surface are reproduced, periodontal reparation, lumbar intervertebral disc degeneration disease, spondylolisthesis disease, osseous tissue are filled.

18. a kind of moldable biomaterial that is prepared by claim 13 or 14 described methods.